Undiagnosed diseases represent a significant burden to patients, their family, and the health care system of the United States. These diseases are hard to manage and even more difficult to treat because their underlying etiology is unknown. The objective of the National Institutes of Health Undiagnosed Disease Program (UDP) is to identify genomic abnormalities that are associated with these diseases. A patient that was part of the UDP program was found to harbor a missense mutation in the FAM109A gene. The patient presented with a range of symptoms including general developmental delay, motor deficit, sensory defects, and abnormal craniofacial and brain development. The goal of this proposal is to determine whether the patient mutation in FAM109A is directly responsible for the observed symptoms. FAM109A has been shown to associate with endocytic vesicles and to function in specific steps of vesicular sorting. However, the role of this gene has not been studied in vivo. In order to examine the causal link between the FAM109A mutation and disease symptoms, we propose to combine molecular studies in cell lines with genetic, physiological, and behavioral studies at the organismal level. The central hypothesis of this proposal is that the mutant FAM109A allele is defective in specific aspects of endocytic sorting, and that these defects directly correlate with patient symptoms. We will compare the ability of wild type versus mutant FAM109A to sustain endocytic sorting in cell culture models. In addition, we will generate a patient-mimetic mutation in the Drosophila and zebrafish homologs of FAM109A using CRISPR-mediated genome editing. Phenotypic analysis of mutant Drosophila and zebrafish will elucidate the in vivo roles of FAM109A and reveal whether the patient mutation is causal to the observed symptoms. Finally, we propose to examine whole genome sequencing databases to determine whether additional patients with developmental disorders harbor mutations in FAM109A.